Mechanisms that create, maintain, and modulate synapses are essential building blocks of human behavior. Disruptions to these mechanisms are inextricably linked to aberrant behavior and diseases ranging from depression and schizophrenia to addiction and Alzheimer?s Disease. Thus, the long-term goal of this grant is to pursue a deep understanding of the molecular organization underlying synaptic transmission and plasticity. Our previous work took advantage of the extremely high-resolution enabled by single-molecule imaging methods and determined that at glutamatergic synapses, key proteins in the active zone and the postsynaptic density are enriched in subsynaptic nanodomains (<100 nm). Most surprisingly, nanodomains of the critical fusion-regulatory proteins RIM and Munc13 in the presynaptic active zone align with high precision across the synaptic cleft from nanodomains enriched in postsynaptic glutamate receptors. Using single-vesicle fusion mapping, we determined that the local density of RIM within active zone subregions predicts the probability of action potential-evoked vesicle fusion. This striking architectural arrangement has important implications for how synapses function. This nano-alignment between release sites and receptors can modulate synaptic transmission and potentially influence intracellular signaling. Preliminary data here and published work from others establishes that transsynaptic nanoalignment is an important element of synaptic architecture, widely present in diverse synapse types. Further, our data provide firm evidence that subsynaptic nanostructure and nanoalignment are dynamically modulated during synaptic plasticity and actively maintained by ongoing molecular interactions. These observations strongly motivate understanding the mechanisms involved in creating and maintaining transsynaptic alignment. Therefore, we will test a set of related but independent hypotheses about the origin and maintenance of transsynaptic nanoalignment. We will test 1) whether two key neurexin partners, neuroligin and LRRTM, cooperate to provide the structural basis of transsynaptic alignment, 2) whether glutamate receptors themselves are necessary or sufficient to influence the nanoscale protein organization of the active zone 3), whether the active zone RIM complex conveys instructive information to establish postsynaptic nanopatterning, and 4) how the actin cytoskeleton exerts ongoing control over synapse nanoscale architecture. To answer these questions, we have worked to establish and apply several new broadly useful technologies. We utilize a new super-resolution imaging methodology to visualize cellular substructure at nanometer resolution in vivo, apply multiplexed single-molecule imaging to map numerous proteins in the same sample, and develop new optical and biochemical tools to acutely control the actin cytoskeleton, adhesion complexes, and receptor distribution with high spatiotemporal resolution and in brain slices. The outcomes of these experiments will answer core questions about the genesis of an important new aspect of synaptic architecture and test the physiological role of synaptic nanoalignment in brain circuits.